Glutamic acid decarboxylase (GAD65) is one of the earliest pancreatic beta-cell antigens that induces a diabetes-related T and B cell autoimmune response. However, recent evidence supports that T cells specific for GAD65 peptides could also function as regulatory agents to inhibit type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Since antigen processing and presentation cells (APC) are the first cellular players to encounter foreign and self antigens, whose interactions are essential for initiating adaptive immune responses and establishing self tolerance, we hypothesize that the existing differences within the antigen processing machinery between T1 D-susceptible and -resistant, MHC-matched individuals are essential for the generation of the pathogenic effectors rather than the regulators, or vice versa. I will study processing and presentation of GAD65 determinants and activation of their specific CD4+ T cells in the susceptible NOD strain and a congenic resistant NOR strain. Two possible, but mutually non-exclusive, processing mechanisms that could affect the choice of peptide determinants from GAD65 to be presented on MHC II molecules will be examined: 1) accessibility to MHC II binding among distant determinants -the exogenous, immunization-induced determinants, likely of high affinity for MHC, vs. the endogenous, spontaneous-arising determinants, that are possibly cryptic;2) differential enzymatic cutting or trimming of flanking amino acid residues of overlapping epitopes within the diabetes-associated determinant regions. T cell lines and hybridoma clones specific for GAD65 peptides will be used to examine the presentation of their specific peptides following processing in the susceptible and resistant animals or in culture by various APC populations. Adoptive transfer experiments will be performed to examine islet infiltration and activation of GAD65-specific T cells that are labeled with dyes and distinguishable surface markers. Most importantly, I will use a powerful repertoire analysis method, CDR3-length spectratyping (Immunoscope), to identify and track the dominant T cell clones in the cell lines, as well as in the islets after their transfer. Finally, I will examine one candidate diabetes-associated locus, Idd5.2, and its candidate gene, Nramp1 (Natural Resistance-Associated Macrophage Protein 1), which are different between the NOD and NOR strains, for their contribution to the apparent abnormal processing and presentation machinery in NOD mice.